Vav guanine nucleotide exchange factors link hyperlipidemia and a prothrombotic state

Abstract

Platelet hyperactivity associated with hyperlipidemia contributes to development of a pro-thrombotic state. We previously showed that oxidized LDL (oxLDL) formed in the setting of hyperlipidemia and atherosclerosis initiated a CD36-mediated signaling cascade leading to platelet hyperactivity. We now show that the guanine nucleotide exchange factors Vav1 and Vav3 were tyrosine phosphorylated in platelets exposed to oxLDL. Pharmacologic inhibition of src family kinases abolished Vav1 phosphorylation by oxLDL in vitro. Coimmunoprecipitations revealed the tyrosine phosphorylated form of src kinase Fyn was associated with Vav1 in platelets exposed to oxLDL. Using a platelet aggregation assay, we demonstrated that Vav1 deficiency, Fyn deficiency, or Vav1/Vav3 deficiency protected mice from diet-induced platelet hyperactivity. Furthermore, flow cytometric analysis revealed that Vav1/Vav3 deficiency significantly inhibited oxLDL-mediated integrin αIIbβIII activation of platelets costimulated with ADP. Finally, we showed with an in vivo carotid artery thrombosis model that genetic deletion of Vav1 and Vav3 together may prevent the development of occlusive thrombi in mice fed a high-fat diet. These findings implicate Vav proteins in oxLDL-mediated platelet activation and suggest that Vav family member(s) may act as critical modulators linking a prothrombotic state and hyperlipidemia.

Figure 1

OxLDL induces phosphorylation of Vav1 and Vav3 in platelets. Washed human platelets (2 × 10⁸/mL) containing 2mM CaCl₂ and 1mM MgCl₂ were incubated with 50 μg/mL native LDL or oxLDL for 30 minutes (A) or 50 μg/mL oxLDL over varying time points (B,D) or various concentrations of oxLDL for 30 minutes (C) and then lysed. Vav1 (A-C) or Vav3 (D) was precipitated by specific Abs and precipitates were analyzed by immunoblot with 4G10 anti-phosphotyrosine Ab. The membranes were then stripped and reprobed with Abs to the total relevant proteins to normalize the protein loaded. Results are representative of at least 3 independent experiments from different donors. nLDL indicates native LDL; and oxLDL, oxidized LDL.

Figure 2

OxLDL-induced Vav1 phosphorylation is mediated by src kinase Fyn. (A) Human platelets were incubated with the broad-spectrum src inhibitor AG1879 before incubation with 50 μg/mL oxLDL. The platelet lysates were then analyzed as in Figure 1 for Vav1 phosphorylation. (B) Platelets were incubated without or with oxLDL and then lysed. Vav1 was precipitated by an Ab specific for Vav1. Precipitates were analyzed by immunoblot with 4G10 anti-phosphotyrosine Ab, Vav1 Ab, and Fyn Ab (mouse). (C) Platelets were incubated without or with oxLDL and then lysed. Fyn was precipitated with an anti-Fyn Ab (mouse). Precipitates were analyzed by immunoblot with Vav1 Ab and Fyn Ab (Rabbit).

Figure 3

High-fat diet-induced platelet hyperreactivity in mice is dependent on Fyn/Vav1 signaling axis. (A) Platelets from WT and vav1^−/− mice on chow or western diet for 2 weeks were stimulated with 2μM ADP. Aggregation was assessed turbidimetrically with a dual-channel aggregometer. Shown are representative curves. (B) Aggregation amplitude of platelets from WT or vav1^−/− mice on chow or western diet or fyn^−/− mice on western diet for 2 weeks incubated with 2 or 5μM ADP as indicated. (C) Platelets from WT and vav1^−/−;Vav3^−/− mice on chow or western diet for 2 weeks were treated with 1μM ADP. Aggregation was assessed turbidimetrically with a dual-channel aggregometer. Aggregation amplitudes were shown as indicated. (D) Platelets from WT and vav1^−/−;Vav3^−/− mice were column purified and incubated with 40 μg/mL native LDL or oxLDL. After 20 minutes, platelets were further incubated with or without 1μM ADP and with JonA Ab for 10 minutes and then fixed before subject to flow cytometric analysis. Percentage increases in MFI of stained platelets treated with ADP and oxLDL in comparison to that of platelets treated with ADP and native LDL were shown.

Figure 4

Vav1 and Vav3 may play redundant but critical roles in in vivo thrombus formation in mice fed high-fat diet. (A) Time to thrombotic occlusion of carotid arteries from vav1^−/− mice and their age-matched wild-type mice fed high-fat diet for 2 weeks was measured after 1 minute topical application of 7.5% FeCl₃. Platelets were labeled by direct injection of fluorescence dye Rhodamine 6G into the jugular vein. Thrombi formation in the carotid artery was assessed under intravital microscopy. (B) Time to thrombotic occlusion of carotid arteries from vav1^−/−;vav3^−/− and their age-matched wild-type mice fed high-fat diet for 4 weeks was measured after 1-minute topical application of 7.5% FeCl₃.